A hierarchical macropore/nano implant surface has been considered as an effective approach to endow biomaterials with better osteogenic performance and long-term stability. The involvement of rho-associated kinase (ROCK) in hierarchical surface-induced osteogenesis, which might originate from the synergistic effect of macropores and nanostructures on cytoskeleton distribution, has previously been demonstrated. However, the underlying intracellular mechanisms are still largely unknown, and thus, this precludes the precise understanding of its cell surface interaction. In this study, it has been further proved that a ROCK-related signaling pathway might be important in translating the surface cues into BMSC fate. Results showed that different bone marrow stromal cell (BMSC) cytoskeleton distributions and corresponding ROCK activity, consequently exerted distinct effects on the FAK-ERK1/2 signaling pathway, which was finally responsible for the BMSC osteogenic differentiation performance. This study has indicated the probable role of ROCK-regulated cytoskeleton tension and its downstream signaling pathway in mediating the signals from topography cues for desirable osteogenic differentiation and furthermore, a probably distinct mechanotransduction process concerning different scales of topographies has been proposed, which might provide insights for use in advanced biomaterial surface design to attain better osteogenesis.